Received: 5 May 2021

| Revised: 2 September 2021 | Accepted: 17 October 2021

DOI: 10.1111/acel.13502  

ORIG INAL PAPER

Sex-­specific accelerated decay in time/activity-­dependent
plasticity and associative memory in an animal model of
Alzheimer's disease
Sheeja Navakkode1,2| Jessica Ruth Gaunt1 | Maria Vazquez Pavon2 |Vibhavari Aysha Bansal1 | Riya Prasad Abraham2 | Yee Song Chong2 |Toh Hean Ch'ng1,3| Sreedharan Sajikumar2,4,5
1
Lee Kong Chian School of Medicine,
Nanyang Technological University,
Singapore, Singapore
2

Department of Physiology, National
University of Singapore, Singapore,
Singapore
3

Abstract
Clinical studies have shown that female brains are more predisposed to neurodegenerative diseases such as Alzheimer's disease (AD), but the cellular and molecular mechanisms behind this disparity remain unknown. In several mouse models of AD, synaptic

School of Biological Science, Nanyang
Technological University, Singapore,
Singaporeplasticity dysfunction is an early event and appears before significant accumulation

4

dimorphism at the synaptic level contributes to the higher risk and prevalence of AD

Healthy Longevity Translational Research
Programme, Yong Loo Lin Schoolof Medicine, National University ofSingapore, Singapore, Singapore
5Life Sciences Institute Neurobiology
Programme, National University ofSingapore, Singapore, Singapore
Correspondence
Toh Hean Ch'ng, Lee Kong Chian Schoolof Medicine, Nanyang Technological
University, Singapore 308232, Singapore.
Email: thchng@ntu.edu.sg
Sreedharan Sajikumar, Departmentof Physiology, National University ofSingapore, Singapore 117597, Singapore.
Email: phssks@nus.edu.sg
Funding information
Singapore Ministry of Education
Academic Research Fund Tier 3, Grant/Award Number: MOE2017-­T3-­1-­0 02;
Nanyang Assistant Professorship(NAP) from Nanyang Technological
University; NUSMED-­FOS Joint Researchof amyloid plaques and neuronal degeneration. However, it is unclear whether sexualin females. Our studies on APP/PS1 (APPSwe/PS1dE9) mouse model show that AD impacts hippocampal long-­term plasticity in a sex-­specific manner. Long-­term potentiation (LTP) induced by strong tetanic stimulation (STET), theta burst stimulation (TBS)and population spike timing-­dependent plasticity (pSTDP) show a faster decay in AD
females compared with age-­matched AD males. In addition, behavioural tagging (BT),a model of associative memory, is specifically impaired in AD females with a fasterdecay in memory compared with males. Together with the plasticity and behaviouraldata, we also observed an upregulation of neuroinflammatory markers, along withdownregulation of transcripts that regulate cellular processes associated with synaptic plasticity and memory in females. Immunohistochemistry of AD brains confirmsthat female APP/PS1 mice carry a higher amyloid plaque burden and have enhanced
microglial activation compared with male APP/PS1 mice. Their presence in the diseased mice also suggests a link between the impairment of LTP and the upregulationof the inflammatory response. Overall, our data show that synaptic plasticity and associative memory impairments are more prominent in females and this might accountfor the faster progression of AD in females.

Abbreviations: AD, Alzheimer’s disease; LTP, Long-­term potentiation; STET, Strong tetanic; TBS, Theta burst Stimulation; BT, Behavioural tagging; pSTDP, population spike timing-­
dependent plasticity; Aβ, Amyloid-­beta; NFTs, neurofibrillary tangles; GWAS, Genome-­wide association studies; aCSF, artificial cerebrospinal fluid; fEPSP, field excitatory postsynaptic
potentials; OF, open field; IA, Inhibitory Avoidance; DEGs, differentially expressed genes; FDR, false discovery rate; GO, gene ontology; PRPs, plasticity-­related proteins; LFCs, log2 foldchanges.
Maria Vazquez Pavon and Vibhavari Aysha Bansal equally contributed to this work.  

This is an open access article under the terms of the Creat​ive Commo​ns Attri​bution License, which permits use, distribution and reproduction in any medium,provided the original work is properly cited.
© 2021 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.
Aging Cell. 2021;20:e13502.
https://doi.org/10.1111/acel.13502

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Programme, Grant/Award Number:
NUHSRO/2018/075/NUSMed-­FoS/01

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NAVAKKODE et al.

KEYWORDS

Alzheimer's disease, behavioural tagging, LTP, sexual dimorphism, STDP, synaptic plasticity,
synaptic tagging and capture, transcriptome profiling

I NTRO D U C TI O N

MacLusky, 2017). Moreover, studies have shown that memory-­
related synaptic plasticity is sexually dimorphic and this is reflected

Alzheimer's disease (AD) is a progressive neurodegenerativein the CA1-­dependent spatial behaviour and predisposition to neu-

disorder characterized by memory loss and behavioural deficits

ropsychiatric disorders such as AD (Wang et al., 2018). Induction(Latimer et al., 2021; Nardini et al., 2021). The hallmarks of AD

of long-­term potentiation (LTP), a cellular correlate of learning andinclude neurodegeneration, the presence of extracellular amyloid-­memory in the CA1 area of hippocampus, was shown to be influ-

beta (Aβ) proteins and intracellular neurofibrillary tangles (NFTs)

enced by the interaction of gonadal hormones (Yang et al., 2004),

made up of abnormally phosphorylated tau protein (Islam Khanand it has been demonstrated that, in female, but not male rodents,

et al., 2018; Mueed et al., 2019; Silva et al., 2019). Although much

LTP and its associated kinases require endogenous expression ofprogress has been made on the molecular basis of AD in the pastoestrogen and its receptor, to determine the threshold for the

few decades, it is still not clear how the confluence of sex dif-induction of LTP and spatial memory (Yang et al., 2004). There is

ferences and other risk factors influences the progression of theevidence that synaptic plasticity is modulated by a number of sex-­

disease. It is well known that AD differentially affects males andspecific signalling mechanisms that vary depending on the brain

females (Dennison et al.,2021). The risk of AD in females is 1 in 6,

region (Hyer et al., 2018). The effects of sexual dimorphism on syn-while in men, it is 1 in 11 (Regitz-­Z agrosek & Seeland, 2012). The

aptic plasticity and memory and its contribution to the higher inci-factors that might contribute to sex differences in AD are diverse

dence of AD in females are not well understood. For example, we

and include differences in genetic background, hormone secre-

still do not fully understand the relationship between sex-­specifiction, activation of microglia and the neuroinflammatory response

plasticity mechanisms and changes in the brain microenvironment

during disease progression (Loeffler, 2021; Mielke, 2018; Watzka

during AD progression, and whether the interaction between these

et al., 1999).

two processes contributes towards the accelerated AD pathology

Genetic variation plays a major role in the relationship between

observed in females.

sex and AD pathology (Breijyeh & Karaman, 2020). Healthy individ-

To understand how synaptic plasticity is involved in the in-uals with a maternal AD history are known to show more prominentcreased vulnerability of females to AD, we examined whether AD

phenotypic changes in vulnerable brain regions compared to thoseaffects the induction and maintenance of different forms of long-­with a paternal AD history (Berti et al., 2011; Mosconi et al., 2010).

lasting LTP in males and females. We specifically examined protein

The ε4 allele of the apolipoprotein E gene is one of the most common

synthesis-­dependent late LTP (L-­LTP) induced by strong tetanus

genetic risk factors for AD (Corder et al., 2004). Genetic variants of(STET), theta burst stimulation (TBS) and population spike timing-­

the apolipoprotein E gene have been shown to confer different risks

dependent plasticity (pSTDP) in both female and male WT and APP/

for male and female AD patients (Corder et al., 2004; Johnson et al.,PS1 mice. We used 4-­ to 5-­month-­old mice for our studies, as this

1998; Payami et al., 1996).

is a time point at which synaptic deficits start to appear but well

Other major contributing factors to sex differences in the pro-before widespread neuronal death (Sadowski et al., 2004; Sun et al.,gression of AD are the differences in microglial activation and el-2019). Behavioural tagging (BT) paradigm was used to test associa-

evation of the neuroinflammatory response (Manji et al., 2019).

tive memory in both sexes from WT and APP/PS1 mice. Our results

Genome-­wide association studies (GWAS) have shown that muta-

show that overall, APP/PS1 mice have impaired L-­LTP and associa-

tions in genes associated with neuroinflammation are a major risk

tive memory, with female APP/PS1 mice showing a faster decay infactor for AD (Zhang et al., 2013). Many of these AD risk factors

plasticity and memory compared with male mice. Transcriptome

converge on the microglia, which suggests that microglial activation

profiling of male and female hippocampus indicated that AD mice,

could be a causal factor for AD (Frigerio et al., 2019). Age-­relatedparticularly female mice, have a robust upregulation of immune-­

expression of hippocampal neuroinflammatory genes is also known

related genes and microglial activation, which was confirmed withto be sexually dimorphic (Mangold et al., 2017).
Along with genetic and microglial influences, structural differ-

immunohistochemistry. In addition, we observed decreased expression of genes associated with neuronal plasticity exclusively

ences at the synapse can also potentially contribute to sex-­specificin female mice. Collectively, our data show that synaptic plasticity

risks of neurodegenerative diseases (Zheng et al., 2019). Reports

impairment is more pronounced in females than in males, and thishave indicated that the CA3 pyramidal cells of males and females

likely contributes to greater cognitive impairments and increased

are distinct in structure, function and plasticity (Scharfman &vulnerability to AD.

| 3 of 21

NAVAKKODE et al.

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M ATE R I A L S A N D M E TH O DS

2.1.3 | Field potential recordings

2.1 | Electrophysiology

In all the electrophysiology recordings, two-­pathway experiments

2.1.1 | Animals

trodes (5 MΩ; AM Systems) were positioned at an adequate distancewere performed. Two monopolar lacquer-­coated stainless steel elecwithin the stratum radiatum of the CA1 region for stimulating two
All animal procedures were approved by the Institutional Animal

independent synaptic inputs S1 and S2 of one neuronal population,

Care and Use Committee (IACUC) of the National University ofthus evoking field excitatory postsynaptic potentials (fEPSP) fromSingapore. We used a mouse model of AD that expresses a mu-

Schaffer collateral/commissural-­C A1 synapses (Figure 1a). Pathway

tated chimeric mouse/human APP and the exon-­9-­deleted variant

specificity was tested using the method described in Sajikumar andof human PS1, both linked to familial AD, under the control of a

Korte (2011). A third electrode (5 MΩ; AM Systems) was placed inprion promoter element (APPSwe/PS1dE9), which we denote as

the CA1 apical dendritic layer for recording fEPSP. After the pre-­

APP/PS1 (Borchelt et al., 1997). We used 4-­  to 5-­month-­old ani-

incubation period, a synaptic input–­output curve (afferent stimula-mals as it is an early stage when synaptic plasticity and behaviouraltion vs. fEPSP slope) was generated. Test stimulation intensity waschanges occur along with Aβ pathology (Gong et al., 2004; Yu et al.,adjusted to elicit fEPSP slope of 40% of the maximal slope response2019). We did not use younger animals as there are mixed reports

for both synaptic inputs S1 and S2. The signals were amplified by aon the plasticity deficits at 2–­3 age group depending on the mousedifferential amplifier, digitized using a CED 1401 analogue-­to-­digitalmodel used (Corder et al., 2004; Trinchese et al., 2004). 70 animals

converter (Cambridge Electronic Design) and monitored online with

were used to isolate 168 slices for electrophysiology experiments

custom-­made software. To induce late LTP, a “strong” tetanizationin this study, out of which 33 were from WT males, 38 from WT(STET) protocol consisting of three trains of 100 pulses at 100 Hz

females, 46 from APP/PS1 males and 51 from APP/PS1 females. For(single burst, stimulus duration of 0.2 ms per polarity), with an inter-behavioural experiments, 56 animals were used with 14 animals intrain interval of 10 min, was used. Theta burst stimulation–­LTP (TBS-­each group. For immunohistochemistry and transcriptome analysis,

LTP) was induced using a protocol, which consisted of 50 bursts

32 animals were used, with eight animals from each group. Thus, a(consisting of four stimuli) at an interstimulus interval of 10 ms. The

total number of 158 animals were used for this study. Animals were

50 bursts were applied over a period of 20 s at 5 Hz.

housed under 12-­h light/12-­h dark conditions with food and water
available ad libitum.

In all experiments, a stable baseline was recorded for at least
30 min using four 0.2-­Hz biphasic constant current pulses (0.1 ms perpolarity) at each time point. Four 0.2-­Hz biphasic, constant current
pulses (spaced at 5 s) given every 5 min were used for postinduction

2.1.2 | Hippocampal slice preparation

recordings, and the average slope values from the four sweeps wereconsidered as one repeat and used for plotting fEPSP percentage vsAnimals were anaesthetized briefly using CO2 and were decapi-

time graphs. Initial slopes of fEPSPs were expressed as percentages

tated, and their brains were quickly removed and transferred toof baseline averages.

4°C artificial cerebrospinal fluid (aCSF)-­a modified Krebs–­R inger
solution containing the following (in mM): 124 NaCl, 3.7 KCl, 1.2
KH2PO 4, 1 MgSO 4 ·7H2O, 2.5 CaCl2·2H2O, 24.6 NaHCO3 and 10

2.2 | Population spike timing-­dependent plasticityd-­g lucose. The pH of aCSF was between 7.3 and 7.4 when bubbled

with 95% oxygen and 5% carbon dioxide (carbogen). Both right

For population spike timing-­dependent plasticity (pSTDP) experi-

and left hippocampi were isolated out in the cold (2–­4°C) aCSF

ments, a stimulating electrode (S0) was positioned in the alveus tobeing continuously bubbled with carbogen (Krishna-­K et al., 2020;

evoke antidromic neuronal action potentials, similar to our earlier

Shetty et al., 2015). Transverse hippocampal slices of 400 μm

report (Pang et al., 2019). The antidromic spikes induced by the

thickness were prepared from the right and left hippocampi using

stimulation of the alveus lead to backpropagating action potentials

a manual tissue chopper (Stoelting), and transferred onto a nylon

within the dendrites of CA1 pyramidal neurons. The alveus stimula-

net in an interface chamber (Scientific Systems Design) and incu-tion regulates the degree of postsynaptic neuronal activity. In addi-bated at 32°C with an aCSF flow rate of 1 ml/min and carbogention to the alveus stimulating electrode, two stimulating electrodes,

consumption of 16 L/h. The entire process of animal dissection,

S1 and S2, were located in the stratum radiatum of the CA1 region

hippocampal slice preparation and placement of slices on theto stimulate Schaffer collateral/commissural fibres. Two record-chamber was done within approximately 5 min to ensure thating electrodes were positioned in the CA1 stratum pyramidale andhippocampal slices were in good condition for electrophysiology

stratum radiatum to record population spikes and field excitatory

studies. The slices were incubated for at least 3 h before start-

postsynaptic potentials (fEPSPs) from the Schaffer collateral/com-

ing the experiments (for more details, see Sajikumar et al., 2005;

missural-­C A1 synapses, respectively (Figure 2a). The test stimula-

Shetty et al., 2015).

tion strength at S1 was set at a subthreshold intensity for population

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F I G U R E 1 APP/PS1 females show a faster decay in L-­LTP induced by STET (a) Schematic representation of the location of electrodesin the CA1 region of a transverse hippocampal slice. Recording electrode (rec) positioned in CA1 apical dendrites was flanked by two
stimulating electrodes S1 and S2 placed in the stratum radiatum (sr) layer to stimulate two independent Schaffer collateral (sc) synaptic
inputs of the same neuronal population. (b) The STET in S1 (blue, filled circles) resulted in a significant potentiation that maintained for 4 h,while the control potentials in S2 (blue, open circles) remained stable throughout the recording in male WT mice (n = 6). (c) STET in S1 (pinkfilled circles) resulted in a long-­lasting LTP for 4 h, while the control input S2 (pink, open circles) was stable for the time course investigatedin female WT mice (n = 7). (d) STET in S1 (blue, filled circles) only resulted in E-­LTP that gradually decayed to baseline in male APP/PS1 mice,(180-­min Wilcox, p = 0.07, U test, p = 0.08; blue, filled circles; n = 7). (e) In female APP/PS1 mice, STET in S1 also resulted in an early form of
LTP, which decayed to baseline (95-­min Wilcox, p = 0.109, U test, p = 0.07; n = 7). Control stimulation of S2 in both d and e showed stablepotentials for the recorded time period (open circles). Error bars in all the graphs indicate ±SEM. Analogue traces represent typical fEPSPs
of inputs S1 and S2, recorded 15 min before (dotted line), 30 min after (dashed line) and 240 min (solid line) after tetanization. Three solid
arrows represent the time of induction of L-­LTP by STET for the induction of late LTP. Scale bars: vertical, 2 mV; horizontal, 3 ms

NAVAKKODE et al.

| 5 of 21

F I G U R E 2 Faster impairment of pSTDP in female APP/PS1 mice with coincidental pre-­ and postsynaptic stimulation. (a) Schematic
diagram of location of electrodes in a hippocampal slice for STDP experiments. The two grey inverted triangles represent recording
electrodes placed in the cell body and dendritic layer of CA1 area hippocampal slices to record population spike and fEPSP, respectively.
Black inverted triangles represent three stimulating electrodes. Stimulating electrodes 1 and 2 (Stim. S1 and Stim. S2) were placed in thestratum radiatum to stimulate two independent SC pathways, and Stim. S0 represents a stimulating electrode located in the alveus layer
to evoke antidromic action potentials in CA1 pyramidal neurons. Analogue traces on the right side represents field potentials that wererecorded from the stratum pyramidale (upper trace) and stratum radiatum (lower trace) in response to coincidental pairing of alveus (Stim.
S0) and stratum radiatum (Stim. S1) stimulation. (b) Simultaneous stimulation of the presynaptic input S1 and the alveus layer (S0; 20
pairings, 1 Hz) induced a persistent pathway-­specific increase in synaptic responses of input S1 (blue, filled circles) that lasted 4 h. Unpaired
input S2 (blue, open circles) remained stable in male WT mice (n = 6). (c) Simultaneous stimulation in female WT slices also resulted in long-­
lasting increase in synaptic responses that lasted for 4 h in paired input S1 (pink, filled circles), while the unpaired control input remained
stable (pink, open circles; n = 10). (d) Pairing at 0 ms in male APP/PS1 mice showed only a decremental LTP (70-­min Wilcox, p = 0.02, U test,
p = 0.1; n = 6). (e) Pairing at 0 ms in female APP/PS1 mice also resulted only in a decremental LTP (40-­min Wilcox, p = 0.2, U test, p = 0.08;
n = 8). In all experiments, the control unpaired input was stable throughout the time period of investigation (open blue and pink circles).
Relative timing Δt represents the time points at which stimulus S0 (postsynaptic component) and S1 (presynaptic component) were initiated.
Solid single arrow represents the time of pairing. Scale bars: vertical, 2 mV; horizontal, 3 ms. Error bars indicate ±SEM. Symbols and analogue
traces as in Figure 1

6 of 21|

spike generation. An input–­output curve was determined for synap-

NAVAKKODE et al.

×35 (length) ×35 cm (height). A weak IA training induces only

tic input S2 (afferent stimulation versus population spike amplitude

short-­term memory, but this can be consolidated to long-­term

or fEPSP). Pathway specificity of two inputs, S1 and S2, was always

memory by the novelty exploration experience, consisting ofdetermined by paired-­pulse stimulations described in Krishna-­K

10 min of OF, that occurs 1 h before IA (Moncada & Viola, 2007;

et al., 2020; Pang et al., 2019. At S2, the test stimulation strength

Wong et al., 2019). The IA apparatus consists of a 50 (width) ×25

was set to produce a population spike amplitude of 40% of the maxi-(height) ×25 cm (length) Plexiglas box with a 5 (height) ×8 (width)

mal response. For the stimulus in S0, the intensity of stimulation was

×25 cm (length) platform on the left end of a series of bars, which

set to elicit an antidromic response of approximately 2 mV in theconstitutes the floor of the box. During the training session, mice

CA1 pyramidal neurons.

were placed on the platform that faces the left rear corner of the

20 pairs of single stimuli (stimulus duration of 0.2 ms/ polarity)box. When they stepped down, putting their four paws on theat 1 Hz were delivered to S0 and S1 at distinct relative time intervals

bars, they received a weak foot shock (0.3 mA, 2 s) after which

for different sets of experiments (Δt = t0-­t1, where t0 and t1 are thethey were removed from the box and returned to their home cage.

times at which stimuli S0 (postsynaptic component) and S1 (presyn-

Memory was evaluated by comparing the step-­d own latency inaptic component) were initiated (Pang et al., 2019).

the training session with that in the test sessions. The cut-­offtime for step-­d own latency was 4 min. A high step-­d own latency

2.3 | Oestrous cycle phaseindicates that the animal has better memory for IA. Step-­d own
latency is represented as percentage values similar to our previous reports (Krishna-­K et al., 2020; Wong et al., 2019). Memory

The oestrous cycle phase of female WT and APP/PS1 mice waswas tested at three different time points: 1, 24 h and 7 days after

identified by using vaginal smears. The vaginal canal opening wastraining sessions. The same animals were used to retest at dif-first rinsed with distilled water, and a mild penetration of vaginal

ferent time points as in previous reports (Krishna-­K et al., 2020;

orifice was done using a pipette and saline (50 µl) was flushed

Wong et al., 2019). As a control experiment, mice received weak

into the vagina gently three to four times with a pipette. The oes-

IA training without OF and memory was evaluated similarly as

trous cycle phase was determined from the vaginal flush using

mentioned above (Figure 3a).

the crystal violet staining as described in McLean et al., 2012.
The vaginal smear was allowed to dry at room temperature. Then,the slide was dipped in crystal violet for 1 min and washed in

2.5 | RNA extraction and sequencing

distilled water for 1 min. This was repeated once more. The oestrous cycle was identified by looking at the variable proportion

Whole right hippocampi were dissected from N = 4 APP/PS1 miceof leukocytes, cornified cells and nucleated epithelial cells. The

and N = 2 wild-­t ype mice of each sex at age 4.5 months, snap-­frozen

smears having predominantly leukocytes were classified as dioes-

and stored at −80°C. Hippocampi were homogenized in TRIzol

trous. Smears having predominantly nucleated epithelial cells andReagent (Invitrogen). Total RNA was then extracted and column-­

very few or no leukocytes were classified as pro-­o estrous and thepurified using the RNeasy kit (Qiagen) according to the user man-smears having mainly cornified cells were classified as oestrous

ual. Directional mRNA libraries were prepared and sequenced by(McLean et al., 2012).

NovogeneAIT Genomics on the Illumina NovaSeq 6000 platform

The levels of gonadal hormones during pro-­o estrous cycle is(45 M 150-­bp paired-­end reads per sample). Prior to sequencing,approximately 60 pg/ml, which is about twice that at the other

RNA quality was assessed by electrophoresis and Agilent 2100

three stages namely oestrus, metoestrus and dioestrus, with

Bioanalyser analysis. All input RNA samples had RIN >8.0.

oestrogen levels of around 33–­3 8 pg/ml (Chen et al., 2009). We
grouped animals into high oestrogen (pro-­o estrus), low oestrogenor non-­p ro-­o estrus, which comprises (oestrus, metoestrus and

2.6 | mRNA sequencing and analyses

dioestrus).
RNAseq data were first processed using Trimmomatic (Bolger et al.,2.4 | Behavioural tagging2014) to trim Illumina adapters and remove low-­quality or short-­
length reads (quality score <15 or length <30 bp). Paired-­end reads
were then aligned to the Genome Reference Consortium mouse ge-

In order to study associative memory, we used a behavioural tag-nome assembly GRCm38 using STAR (Dobin et al., 2013) and quanti-ging (BT) paradigm (Moncada & Viola, 2007; Wong et al., 2019).

fied by HTseq (Anders et al., 2015) on the Gekko high-­performance

Male and female WT and APP/PS1 mice were used for this study.

computing cluster at Nanyang Technological University. Counts

They were habituated to the room, 12 h before experiments. The

were filtered to retain genes with at least one log2-­transformed

mice were placed in an open field (OF) for 10 min. 1 h after OF, in-count per million mapped reads (log-­CPMs) in a minimum of threehibitory avoidance (IA) training was given to the mice (Figure 3b).

samples (N = 15,521), and upper-­quartile normalization was applied.

The OF consists of a plastic box with dimensions of 35 (width)

Differential expression (DE) analysis was conducted using edgeR

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NAVAKKODE et al.

F I G U R E 3 Impairment of associative memory in female APP/PS1 mice. (a) Schematic diagram of the experimental protocol used forcontrol BT paradigm. A mouse was given weak IA training by providing a weak foot shock consisting of 0.3 mA for 2 s. Step-­down latency
was tested at 1, 24 h and 7 days post-­IA. Memory for IA learning was observed only at 1 h in all four groups: WT males (filled blue bars);
APP/PS1 males (open blue bars); WT females (filled pink bars); and APP/PS1 females (pink open bars; n = 7 from all groups). (b) Schematic
diagram of the experimental protocol used for BT paradigm. Mice were given weak IA training, 1 h after exposure to a novel environment(open field, OF) for 10 min. Step-­down latency was tested at 1, 24 h and 7 days post-­IA. Associative memory was observed in both WT
males and females (filled blue and pink bars, respectively) and in APP/PS1 males (open blue bars) at 24 h, while in APP/PS1 females (openpink bars), associative memory was impaired (n = 7 from all groups). Statistical analyses for latencies at each time point (1, 24 h and 7 days)
for all groups were compared against their training period (asterisks in pink). Further statistical tests were performed between WT and
APP/PS1 animals (black asterisks), as well as between male and female APP/PS1 mice (green asterisks). Error bars indicate ±SEM. Asterisks
indicate significant differences between groups (ns, not significant, **p < 0.01, ***p < 0.001 and ****p < 0.0001)(likelihood ratio test with robust dispersion estimation; Robinson

in each term with LFCs above and below 0 [up-­ and downregulated,

et al., 2010). To reduce the impact of unwanted variation, we used

respectively]). For genes in the GO term “regulation of synaptic

RUVseq (Risso et al., 2014) to identify factors of variation in theplasticity” (GO:0048167), we also plotted rankings by FDR along-expression of empirically defined negative control genes (all genes

side Gaussian kernel density estimation to compare the probabilities

except the top-­ranked 5000 in a first-­pass differential expressionthat these genes were upregulated and downregulated in AD miceanalysis) and we incorporated the top two factors into the model forof each sex. To identify the cell types in which the genes of interest

DE analysis. We analysed differential expression between AD and

are most highly expressed, we used the transcriptome database of

WT conditions for each sex and both combined, as well as the inter-mouse cortical cell types by (Zhang et al., 2014). Analyses were con-action effect defined as [(AD.F -­ WT.F) -­ (AD.M -­ WT.M)]. We identi-

ducted in R (v4.0.2, R Core Team, 2020), and figures were generated

fied DE genes using a false discovery rate (FDR) threshold of 10%.

using ggplot2 (Wickham, 2016).

Hierarchical clustering of DEGs between AP and WT mice was conducted on batch-­corrected CPMs using the Pearson distances withthe ward. D2 algorithm. To analyse enriched biological functions,

2.7 | Immunohistochemistry

we conducted Kolmogorov–­Smirnov tests for GO Biological Process
terms on genes ranked by FDR from DE tests using the topGO pack-Hippocampal sections were prepared using a vibratome (4°C at

age (Alexa & Rahnenführer, 2020), using the weight01 algorithm to

100 µm). Sections that came off the vibratome were immediately

reduce redundancy between terms (Alexa et al., 2006). To determine

fixed with 4% PFA in PBS overnight at 4°C, washed and kept in cry-whether gene sets were predominantly upregulated or downregu-

oprotectant (30% ethylene glycol, 30% glycerol and 10% 0.2 M PB

lated compared with the control groups, we then calculated direc-

in Milli-­Q ) at −80°C. Prior to processing, sections were washed 3X

tion scores for each term (formula: (ΣNup -­  ΣNdown)/√(ΣNup +in PBS and permeabilized in 0.1% Triton X in PBS before antigen re-ΣNdown), where Nup and Ndown represent the number of genes

trieval (10 mM Tris Base, 1 mM EDTA and 10% Triton X in Milli-­Q at

8 of 21|37°C for 15 min). Sections were washed (5X, 5 min) in 0.1% Triton

NAVAKKODE et al.

prolonged recordings (Pang et al., 2019; Sajikumar & Korte, 2011). “n”

X in 1X PBS and blocked for 1 h (10% goat serum, 0.1% Triton X

represents the number of slices in in vitro electrophysiology or num-in PBS) before incubation with primary antibodies (overnight) fol-ber of animals in behavioural experiments. Slices from a minimum of

lowed by secondary antibodies (4 h) with washes (3X, 0.1% Triton

3–­4 biological replicates were used for all in vitro and biochemistry

X in PBS) in between incubations. All sections were mounted on

experiments. Statistical comparisons for behavioural tagging wereslides using Aqua-­Poly/Mount. Unless otherwise stated, all proto-

performed using unpaired Student's t test or one-­way ANOVA (para-cols were performed at room temperature with gentle rocking withmetric) (Krishna-­K et al., 2020; Wong et al., 2019). p < 0.05 was con-an orbital shaker. Antibodies and dyes used include the following:

sidered as the cut-­off for statistically significant differences.

mouse anti-­b eta amyloid antibody (MOAB-­2; Abcam at 1:1000 dilution), mouse anti-­NeuN (Merck-­Millipore at 1:1000), rabbit anti-­
Iba-­1 (Wako at 1:1000), rat anti-­FcγRIIb (R&D Systems at 1:500),
Alexa Fluor-­conjugated secondary antibodies (ABLife Tech. at

3|

R E S U LT S

1:1000) and Hoechst 33342 (Thermo Fisher at 1:1000).

3.1 | Faster decay of STET-­induced L-­LTP in female
APP/PS1 mice

2.8 | Image acquisition and analysis

In order to study long-­term potentiation (LTP) in APP/PS1 mice,
we applied STET in four groups of animals: WT males and females,

All widefield images were acquired using Zeiss Axio Scan.Z1 with

APP/PS1 males and females. In both male and female WT animals,

a 10X objective with a Hamamatsu Orca Flash detector. Magnified

STET application in synaptic input S1 resulted in L-­LTP that lasted

images were taken using Zeiss LSM800 inverted scanning confocal

for 240 min (Figure 1b,c; Figure 1b, n = 6, 1-­min Wilcox, p = 0.03,

microscope using either 40X or 63X plan Apochromat 1.4NA oil im-

1-­min U test, p = 0.002, 240-­min Wilcox, p = 0.03, 240-­min U test,

mersion objectives. For all widefield images, quantification was done

p = 0.002; Figure 1c; n = 7, 1-­min Wilcox, p = 0.02, 1-­min U test,

using IMARIS x64 9.6.0. The hippocampal region was first defined by

p = 0.0006, 240-­min Wilcox, p = 0.02, 240-­min U test, p = 0.005).

drawing a boundary based on organization of the distinct cell body

We did not find a statistically significant difference in the potentia-

layers guided by Hoechst nuclear dye labelling. An intensity thresh-tion between WT males and females at any compared time points (Uold (lower 10% signal) was employed for all antibodies to remove

test, p > 0.05; Figure 1b,c). However, in both male and female APP/

noise. A size-­exclusion filter based on signal intensity was then used

PS1 mice, the same experimental design yielded a decaying form of

to accurately select substructures within each slice and to exclude

LTP with an observable faster decay of potentiation in females com-any staining artefacts (MOAB-­2: 10 µm2; Iba-­1: 3 µm2; NeuN: 3 µm2).

pared with males (Figure 1d,e). STET resulted in an E-­LTP that lasted

The total count per slice, average intensity and area for each sub-for 180 min in AD males (Figure 1d, n = 7, Wilcox, p = 0.07, U test,

structure were compiled, analysed and graphed as appropriate. For

p = 0.08; 1-­min Wilcox, p = 0.008, 1-­min U test, p = 0.0002, 240-­

plaque burden and microglia count, the total counts for each slice

min Wilcox, p = 0.25, 240-­min U test, p = 0.279). However, LTP inwere normalized against area measured. Average FcγRIIb signal for

AD females resulted in an E-­LTP that lasted only 95 min (Figure 1e,

microglia and neurons were calculated based on colocalization ofn = 7, Wilcox, p = 0.109, U test, p = 0.07; 1-­min Wilcox, p = 0.008,

signal with Iba-­1-­ and NeuN-­positive structures, respectively. Unless

1-­min U test, p = 0.0002, 240-­min Wilcox, p = 0.469, 240-­min U

otherwise stated, all graphs and statistical analyses were generated

test, p = 0.13). The faster decay of LTP in females was significant

using GraphPad Prism (v. 8.4.3).

from 30 min (Figure 1d,e; 30-­min U test, p = 0.02). Control baseline stimulation of input S2 in all groups revealed stable potentials

2.9 | Statistics

throughout the entire recording time period of 240 min. Overall, ourresults showed faster decay of STET-­induced L-­LTP in APP/PS1 females compared with males.

In field electrophysiological recordings, the strength of the synaptic
responses was measured as the slope of fEPSP (millivolts per millisecond). All data are represented as mean ± SEM. To test for statisticalsignificance within group, the Wilcoxon signed-­rank test (represented

3.2 | Faster decay of TBS-­induced L-­LTP in female
APP/PS1 miceas Wilcox) was applied to compare the mean normalized fEPSPs at
specified time points with the fEPSP at −15 min (taken as the baseline

Next, we examined theta burst stimulation (TBS) induced L-­LTP

fEPSP). To compare between different groups, the Mann–­Whitney U

using 50 bursts at 5 Hz. TBS resulted in a robust L-­LTP in bothtest (represented as U test) was used. Differences were consideredmale and female WT animals (Figure 4a,b), which lasted foras statistically significant when p < 0.05. Nonparametric tests were

240 min (Figure 4a, n = 7,1-­min Wilcox, p = 0.016, 1-­min U test,

selected because a Gaussian normal distribution could not always

p = 0.0006, 240-­min Wilcox, p = 0.03, 240-­min U test, p = 0.002;

be assumed due to the small sample size per series and analyses of

Figure 4b; n = 7, 1-­min Wilcox, p = 0.01, 1-­min U test, p = 0.0006,

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| 9 of 21

F I G U R E 4 APP/PS1 females show a faster decay in TBS-­LTP. (a) Theta burst stimulation resulted in an increase in synaptic response
in S1 (blue, filled circles) that lasted for 4 h, while the control input S2 (blue, open circles) remained stable for 4 h in male WT mice (n = 7).
(b) Late-­ LTP was maintained for 4 h, when TBS was applied to S1 (pink filled circles) in female WT mice (n = 7). (c) TBS applied to S1 (blue,
filled circles) resulted only in an E-­LTP that gradually decayed to baseline in male APP/PS1 mice (90-­min Wilcox, p = 0.079, U test, p = 0.05;
n = 7). (d) TBS was delivered to S1 (pink, filled circles) in female APP/PS1 mice again showed only an early form of LTP (70-­min Wilcox test,
p = 0.084, U test, p = 0.1; n = 10). Control inputs in all remained stable (open circles). Solid single arrow represents the time of induction of
TBS stimulation in S1. Scale bars: vertical, 2 mV; horizontal, 3 ms. Error bars indicate ±SEM. Symbols and analogue traces as in Figure 1

240-­min Wilcox, p = 0.015, 240-­min U test, p = 0.0006). We did
not observe any difference in the amplitude of LTP between male

3.3 | Faster decay of population spike timing-­
dependent plasticity in female APP/PS1 miceand female WT animals at any compared time points (Figure 4a,b,p > 0.05). As expected, we saw an impairment in TBS-­LTP in both

Spike timing-­dependent plasticity is a phenomenon where the ordermale and female APP/PS1 mice (Figure 4c,d). TBS resulted in an

and precise timing of spikes determine the direction and magnitude

E-­LTP, and the potentiation was significant until 90 min in malesof plasticity and is often considered as the first law of synaptic plas-(Wilcox, p = 0.079, U test, p = 0.05; Figure 4c; n = 7, 1-­min Wilcox,

ticity (Dan & Poo, 2006; Feldman, 2012). Compared with STET and

p = 0.016, 1-­min U test, p = 0.0006, 240-­min Wilcox, p = 0.2188,

TBS, where the stimulus is delivered to the entire presynaptic axon,

240-­min U test, p = 0.2403). In females, the potentiation re-

the minimal nature of STDP protocols, which requires only pairing of

mained significant only until 70 min (Wilcox test, p = 0.084, U

spikes, makes it a comprehensive learning rule for synapses (Shouval

test, p = 0.1; Figure 4d, n = 10, 1-­min Wilcox, p = 0.002, 1-­minet al., 2010). We have reported earlier that coincidental pre-­  andU test, p = <0.0001, 240-­min Wilcox, p = 0.47, 240-­min U test,

postsynaptic stimulation induces persistent potentiation in the CA1

p = 0.72). Interestingly, we also observed a difference in the in-area of rat hippocampal slices (Pang et al., 2019). Thus, we firstduction of TBS-­LTP in both sexes of APP/PS1 mice (Figure 4c,d;tested pSTDP by pairing pre-­ and postsynaptic inputs at 0 ms (S0;5-­min U test, p = 0.02). Overall, our results showed reduced induc-relative timing interval between S0 and S1 stimulations Δt = 0 ms)

tion and faster decay of TBS-­induced L-­LTP in APP/PS1 females as

in male and female WT mice (Figure 2b,c). Long-­lasting potentia-

compared to males.

tion was observed until 240 min in both male and female WT mice

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NAVAKKODE et al.

(Figure 2b, n = 6, 5-­min Wilcox, p = 0.03, 5-­min U test, p = 0.002,

p = 0.1), while in females, it lasted only until 55 min (Figure 5d, n = 6,240-­min Wilcox, p = 0.03, 240-­min U test, p = 0.002; Figure 2c,

Wilcox, p = 0.94, U test, p = 0.09).

n = 10, 5-­min Wilcox, p = 0.002, 5-­min U test, p = <0.0001, 240-­min
Wilcox, p = 0.009, 240-­min U test, p = 0.007).

Next, we explored whether backward pairing with a time interval of 10 ms has any effect on the persistence of potentiation

Next, we repeated the same experiments in APP/PS1 mice. As(Δt = −10 ms). Similar to Figure 5a,b, we observed a long-­lasting

expected, pSTDP in both male and female mice was impaired in

pSTDP in both WT males and females that lasted 240 min (Figure 5e,f;pSTDP (Figure 2d,e) resulting only in an E-­LTP. Statistically signifi-

Figure 5e, n = 7, 5-­min Wilcox, p = 0.03, U test, p = 0.002, 240-­mincant pSTDP maintained until 70 min in males (Wilcox, p = 0.02, U

Wilcox, p = 0.04, U test, p = 0.04; Figure 5f, n = 7, 5-­min Wilcox,

test, p = 0.1), and only until 40 min in females (Wilcox, p = 0.2, U

p = 0.04, U test, p = 0.002, 240-­min Wilcox, p = 0.03, U test,

test, p = 0.0830; Figure 2d; n = 6, 5-­min Wilcox, p = 0.03, 5-­min U

p = 0.002). However, when the same experiments were repeated in

test, p = 0.002, 240-­min Wilcox, p = 0.6, 240-­min U test, p = 0.4;

APP/PS1 mice, the pSTDP was impaired in both males and females

Figure 2e; n = 8, 5-­min Wilcox, p = 0.016, 5-­min U test, p = 0.001,

resulting only in a short-­term potentiation (STP; Figure 5g,h). In APP/

240-­min Wilcox test, p = 0.2, 240-­min U test, p = 0.5). Significant

PS1 males, statistically significant potentiation was maintained until

differences in the amplitude of potentiation in pSTDP were ob-

20 min (Figure 5g, n = Wilcox, p = 0.08, U test, p = 0.1), while inserved between APP/PS1 males and females at 5 min and 120 min

females, potentiation sustained only until 15 min (Figure 5h, n = 6,

(Figures 2d,e, 5-­min U test, p = 0.02, 120-­min U test, p = 0.04), sug-

Wilcox, p = 0.06, U test, p = 0.06).

gesting that the decay of timing-­induced plasticity at 0 ms was fasterin females than in males.

3.4 | Impaired STDP in APP/PS1 mice withforward and backward pairing of synaptic activity at
10 ms

3.5 | Associative memory showed higher decline infemales than in males
In order to study whether the synaptic plasticity deficits observedin hippocampal slices were reflected in hippocampus-­dependent
memory tasks, we studied associative memory in WT and APP/PS1

Next, we wanted to examine whether the persistence of plasticity is

mice using the behavioural tagging (BT) paradigm (Moncada & Viola,affected in APP/PS1 mice by changing the timing and order of pre-­ 2007; Wong et al., 2019). In BT, spatial novelty in the form of an open

and postsynaptic activity. Forward pairing of pre-­  and postsynap-

field (OF) induces the synthesis of plasticity-­related proteins that will

tic stimulations at a positive time interval of 10 ms (Δt = +10) in

be captured by a subsequent weak stimulus in the form of a mild footWT male and female mice resulted in persistent pSTDP that lasted

shock (weak memory), thus allowing weaker memories to be con-

240 min (Figure 5a,b; Figure 5a, n = 7, 5-­min Wilcox, p = 0.0156,

solidated into long-­term memories according to the synaptic tagging

U test, p = 0.006, 240-­min Wilcox, p = 0.02, U test, p = 0.006;

and capture hypothesis (Moncada & Viola, 2007; Shetty & Sajikumar,

Figure 5b, n = 7, 5-­min Wilcox, p = 0.02, U test, p = 0.01, 240-­min2017). It has been demonstrated earlier that a weak memory can be

Wilcox, p = 0.02, U test, p = 0.02). The potentiation observed atconsolidated into a strong memory, if it occurs in close associationΔt = 10 ms in Figure 5a,b was similar to the one elicited by simulta-

with a strong stimulus (OF) (Moncada & Viola, 2007). Memory was

neous pre-­ and postsynaptic stimulations as in Figure 2b,c. However,measured as the latency to step down onto the bars, and a longer

when the same set of experiments was repeated in male and fe-

step-­down latency indicates a stronger memory association. The

male APP/PS1 mice, we observed a decremental pSTDP (E-­LTP;

time points are typically used to assess short-­term memory (STM:

Figure 5c,d). In male APP/PS1 mice, the synaptic potentials remained

1 h after the training session), long-­term memory (LTM: 24 h after

significant until 60 min (Figure 2c, n = 6, Wilcox, p = 0.63, U test,

training) and remote LTM (7 days after training), respectively.

F I G U R E 5 Synaptic rules for STDP are altered in APP/PS1 mice. (a) Forward pairing of pre-­ and postsynaptic stimulations at positive timeinterval of 10 ms led to persistent potentiation of synaptic responses in the synaptic input S1 (blue, filled circles) in male WT mice (n = 7).
(b) Persistent potentiation of 4 h was observed in the synaptic input S1 (pink, filled circles) using the same experimental paradigm as in A infemale WT mice (n = 7). (c) Pairing of pre-­ and postsynaptic inputs as in A and B in male APP/PS1 mice resulted only in a short-­lasting LTP in
S1 (blue, filled circles; 60-­min Wilcox, p = 0.63, U test, p = 0.1; n = 6). (d) Pairing at positive time interval of 10 ms in female APP/PS1 mice
resulted only in a short-­lasting potentiation in S1 (pink, filled circles; 55-­min Wilcox, p = 0.94, U test, p = 0.09; n = 6). (e) Backward pairing
or repeated pairing (arrow) of alveus stimulation (S0) with subsequent Schaffer collateral stimulation in S1 (blue, filled circles) with a relative
time lag of −10 ms resulted in a persistent potentiation lasting 4 h in S1 (blue, filled circles) in male WT mice. (n = 7). (f) Same experiment was
repeated in female WT, which also resulted in a persistent potentiation lasting 4 h in S1 (pink, filled circles), (n = 7). (g) Backward pairing at−10 ms in male APP/PS1 mice resulted in a decremental LTP (blue, filled circles) in S1 (20-­min, Wilcox, p = 0.08, U test, p = 0.1; n = 7). (h) In
female APP/PS1 mice also, only a decremental LTP was observed in S1 (pink, filled circles) after backward pairing at time interval of 10 ms(15-­min Wilcox, p = 0.06, U test, p = 0.06; n = 7). Solid single arrow represents the time of pairing. Scale bars: vertical, 2 mV; horizontal,
3 ms. Error bars indicate ±SEM. Symbols and analogue traces as in Figure 1

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12 of 21|

In the control group (weak IA alone), four sets of animals (WT

NAVAKKODE et al.

Our results indicate that exposing mice to OF prior to weak IA

male and female and APP/PS1 male and female) were subjected to a

stimuli enhanced learning across all time points for both WT males

weak foot shock and tested for inhibitory avoidance (IA) by measur-and females. However, in APP/PS1 males, STM and LTM remained

ing step-­down latency at various time points after training (1, 24 h,

intact while remote memory was abolished. In contrast, female APP/

7 days; Figure 3a). In the experimental BT group (OF before weakPS1 mice showed STM but LTM and remote memory were abolished.

IA), another four sets of mice were subjected to the same conditions

Thus, female APP/PS1 are able to acquire IA memory, while theyas the control group, except that they were first exposed to OF forshow impairment in the formation of IA-­LTM.

10 min prior to the application of a weak foot shock (Figure 3b).
In the control group, when latency for training was comparedwith latency for test sessions, significant difference was observed at
1 h, but not at 24 h and 7 days in WT and APP/PS1 males (WT males;
Figure 3a, blue solid bars, 1 h, p = <0.0001; 24 h, p = 0.06; 7 days,

3.6 | Activation of neuroinflammatory genes anddownregulation of plasticity-­related genes in APP/
PS1 female micep = 0.3; APP/PS1 males; Figure 3a, open blue bars, 1 h, p = <0.0001;
24 h, p = 0.09; 7 days, p = 0.9). Similar findings were observed in

We investigated gene expression in the hippocampus of 4-­  to

WT and APP/PS1 females (WT females; Figure 3a, solid pink bars,

5-­month-­old APP/PS1 and WT subjects of both sexes using RNAseq.

1 h, p = <0.0001; 24 h, p = 0.2; 7 days, p = 0.7; APP/PS1 females;

We detected 323 and 152 differentially expressed genes (DEGs) in

Figure 3a, open pink bars, 1 h, p = 0.003; 24 h, p = 0.08; 7 days,

female and male APP/PS1 mice, respectively, as compared to WT

p = 0.6). Our results show that mild foot shock induces STM but not(with an overlap of 92 DEGs; FDR <0.1), indicating that there is a

LTM or remote memories in all groups of mice.

greater effect of AD pathology on gene expression in female mice

Notably, while both APP/PS1 males and females recalled weakat this age (Figure 6a,b and Figure S1a). For both sexes, the top up-

IA learning at 1 h, the mutant mice still exhibit significant impair-regulated genes are similar and associated with the immune system:

ment when compared to their WT counterparts (male WT [solid blue

Thy1, Ccl6, Itgax, Clec7a and Cst7.

bars] vs male APP/PS1 [open blue bars] at 1 h, U test, p = 0.0023;

To investigate biological processes that may be affected

female WT [solid pink bars] vs female APP/PS1 [open pink bars], U

by the differential transcriptional response between maletest, p = 0.0022).

and female mice, we performed gene ontology (GO) analysis

In the BT group, where OF preceded weak IA, both WT males

using Kolmogorov–­S mirnov tests on genes ranked by FDR.

and females showed STM (1 h), LTM (24 h) and remote memory

As this method does not require a DE threshold, results canlasting 7 days (Figure 3b, solid blue bars and solid pink bars, re-be directly compared between sexes. We found that the top

spectively; WT males; 1 h, p = <0.0001; 24 h, p = <0.0001; 7 days,

enriched terms in female mice indicated robust activation of

p = <0.0001; WT females; 1 h, p = <0.0001; 24 h, p = <0.0001;

the immune system, including increased cytokine production,

7 days, p = <0.0001). The APP/PS1 males showed LTM at 24 h, but

and activation of microglia and innate inflammatory responses

not remote memory at 7 days (APP/PS1 males; open blue bars, 1 h,

(Figure 6ci). The upregulated inflammatory genes include

p = 0.003; 24 h, p = 0.004; 7 days, p = 0.1), whereas in APP/PS1

Cst7, Tyrobp, Trem2, Tlr2, Ctss and members of the Naip family.

females, both LTM and remote memory were abolished (APP/PS1

Similar to female mice, immune genes were also significantly

females; 1 h, p = 0.003; 24 h, p = 0.3; 7 days, p = 0.98).

upregulated in male mice (Figure 6ci) although the top en-

APP/PS1 mice in the BT group showed a deficit in learning rel-riched terms in male mice were also linked to a range of otherative to WT mice of the same sex at all time points (Figure 3b, WT

processes including G protein-­c oupled receptor signalling andmale [solid blue bars] vs APP/PS1 males [open blue bars] at 1 h, U

cellular adhesion (Figure S1b). Another GO term strongly en-

test, p = 0.001; WT female [solid pink bars] vs APP/PS1 females atriched in both sexes is related to cholesterol biosynthesis, a

1 h, U test, p = 0.0007). When latency was compared at 24 h andprocess that has been reported to be widely dysregulated in

7 days post-­training, similar results were observed (24 h, WT male

AD (Sun et al., 2015).

vs APP/PS1 males, U test, p = 0.0003, WT female vs APP/PS1 fe-

While the majority of GO terms described upregulated setsmale, U test, p = 0.0007); 7 days post-­training, WT male vs APP/PS1

of genes, it is notable that the downregulated gene sets (direc-

males, U test, p = 0.0003, WT female vs APP/PS1 female, U test,tion score [DS] <0) in female mice were primarily associatedp = 0.0007).

with memory and plasticity, including regulation of calcium re-In addition, the BT paradigm also uncovered subtle sex-­specificlease and transport, and regulation of the development of den-differences at 24 h and 7 days in APP/PS1 mice, when compareddritic spines and neuronal projections (Figure 6cii). These gene

between the groups (APP/PS1 males vs APP/PS1 females; 24 h, Usets were not downregulated in male APP/PS1 mice (Figure 6cii).

test, p = 0.009; 7 days, U test, p = 0.007; in the control group, maleTo extend this analysis, we examined the differential expres-

APP/PS1 [open blue bars] vs female APP/PS1 [open pink bars] at 1 h,sion of all genes annotated to the GO term “regulation of syn-U test, p = 0.0076; Figure 3a). Comparison of step-­down latency be-

aptic plasticity” (n = 208) and found that these genes were both

tween WT males and females did not show significant difference atsignificantly altered and predominantly downregulated in fe-

any points.

males (P = 1.3 × 10 −5, DS = −4.02), but not in males (p = 0.016,

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| 13 of 21

F I G U R E 6 APP/PS1 female hippocampus is enriched in neuroinflammatory genes and depleted in genes associated with neuronalplasticity and memory. (a) Heatmap showing differentially expressed genes (DEGs) between APP/PS1 (AD) and wild-­t ype (WT) mice(n = 445; FDR <0.1). (b) Volcano plots showing log2 fold changes (LFCs) compared with p-­values from AD vs WT differential expression tests
for female and male mice. (c) Enriched Gene Ontology terms sorted by p-­value in females (most significantly enriched at the top). Direction
scores (DS) indicate overall up-­ or downregulation of genes in each term (abbreviations: +ve, positive; -­ve, negative; reg., regulation; LPS,
lipopolysaccharide; dev., development; EPSP, excitatory postsynaptic potential; Ca, calcium; loc., localization). (ci) Top enriched terms infemales. (cii) Selected downregulated enriched terms associated with memory and plasticity (9/15 top terms with DS<0; p < 0.01). (d) Genes
associated with the GO term “regulation of synaptic plasticity” ranked by FDR in DE tests comparing genotypes for each sex. Genes withhigher expression in AD mice are indicated in red, and lower in blue. Density plots represent the probability that plasticity genes are found ateach point in the ranked list. (e) Overlap between DEGs in male and female AD vs WT mice (FDR <0.1) and genes with a potential interaction
effect (p < 0.05), indicating significantly different effects of genotype between sexes. The N = 106 genes overlapping between groups aresex-­regulated DEGs. (f) Heatmap of the sex-­regulated DEGs identified in 6E. Plot shows LFCs for females (AD v WT), males (AD v WT) andthe interaction effect. A positive interaction effect indicates that a gene is more highly expressed in female than in male AD mice relative
to WT mice of same sex, that is, upregulated in females or downregulated in males. For each gene, the cell type (neuron, microglia, or other)with highest expression in the transcriptome database of (Zhang et al., 2014) is indicated above the plot

DS = −0.14; Figure 6d). In summary, while immune genes are up-

We next identified genes that were affected differently by APP/

regulated in both sexes, plasticity-­r elated genes are mainly down-

PS1 pathology in male and female mice. Of the protein-­coding genes

regulated in female APP/PS1 mice.

that were differentially expressed between genotypes in either

14 of 21|

or both sexes, 106 had an interaction effect with a P-­value <0.05

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4|

DISCUSSION(Figure 6e). Approximately three quarters of these sex-­regulated
DEGs were differentially expressed only in females (n = 75) withIn AD, females are disproportionately affected in terms of enhanced

most of them upregulated (n = 58; Figure 6e,f). Once again, con-pathology, more severe cognitive decline and other clinical manifes-

sistent with our overall observations, many of these female-­specifictations during the progression of the disease (Arnsten et al., 2021;

DEGs are highly expressed in microglia and involved in immune andKoran et al., 2017; Ward et al., 2012). In agreement with this observa-

inflammatory processes (Figure 6f). Notably, some of the female-­tion, our results in an animal model of AD showed that both activity

specific DEGs are expressed in neurons and implicated in AD, includ-and time-­dependent plasticity decline faster in females as compareding genes known to regulate synaptic plasticity; in particular, Mef2c

to males. Our behavioural assay using the BT paradigm showed that(transcription factor), Sema3a (axon guidance), FcγRIIb (Ig receptor)

overall, APP/PS1 mice have strong deficits in memory formation at

and genes reportedly involved in AMPAR trafficking, Syt17, Nptx1

all time points tested. Additionally, the assay revealed that female

and Myo5b (Figure S1c) (Figueiro-­Silva et al., 2015; Kam et al., 2013;

APP/PS1 mice have slightly more robust deficits in LTM and forma-

Ruhl et al., 2019; Sao et al., 2018; Tansey et al., 2018; Wang et al.,tion of associative memories as compared to the male APP/PS1 mice.

2008).

Since LTP remains a prominent cellular model for the persistence oflong-­term memories, a more rapid decay of LTP in female APP/PS1

3.7 | APP/PS1 female mice have more Aβplaques and Iba-­1-­positive microglia in the
hippocampus

mice may be associated with a more severe cognitive impairment
or memory loss in female AD patients (Barnes et al., 2005; Cohenet al., 1993; Ott et al., 1996). In trying to elucidate the molecularbasis for the sex-­specific differences in long-­term plasticity, our transcriptome analysis revealed that APP/PS1 mice exhibit a strong neu-

Our transcriptome analysis indicates a robust upregulation ofroinflammatory response and enhanced microglial activation along

neuroinflammatory and microglial genes that could serve as awith an increase in Aβ pathology. We also found that plasticity genes

molecular basis for the greater impairment in long-­term plastic-

were downregulated in female compared with male APP/PS1 mice,

ity in female AD mice. To verify that the immune system is dif-suggesting that the accelerated Aβ pathology in females likely dis-

ferentially activated in male and female mice, we sectioned therupts the expression of plasticity-­related proteins (PRPs), leading tohippocampus of AD mice from both sexes (4–­5 months) and per-the accelerated decay of LTP and memory in females compared withformed immunohistochemistry using antibodies targeted against

males. The downregulation of PRPs could be due to alterations in

Aβ (MOAB-­2) and microglia (Iba-­1). Quantification of Aβ plaques

the function of NMDA and AMPA receptors, as sex differences are

and Iba-­1-­p ositive microglia indicated that female APP/PS1 micefound in synaptic glutamate signalling (Mota et al., 2014; Qu et al.,have an accelerated Aβ pathology with an increase not only in

2020; Wickens et al., 2018). An increase in glutamate levels severely

plaque burden and plaque size but also in Iba-­1-­p ositive microglia

affects AD males compared with females as they exhibit lower levelsin the hippocampus (Figure 7a-­e).

of GluA2-­containing AMPA receptor subunits (Wickens et al., 2018).

FcγRIIb encodes an Fc receptor that is upregulated in activatedThus, our findings support the observation that females are more

microglia in response to brain injury (Quan et al., 2009). In our tran-

vulnerable to synaptic and memory dysfunctions during progression

scriptome profile, FcγRIIb expression showed sex-­specific differ-of the disease.

ences via mRNAseq and we immunostained for FcγRIIb protein in

Sex differences in neuronal plasticity have been described.

hippocampal slices to examine the expression pattern of the protein

Rodents are reported to have sex differences in spine density acrossin neurons and microglia. Our imaging results detected a significant

different brain regions (Forlano & Woolley, 2010; Gould et al., 1990;

increase in FcγRIIb protein expression in female APP/PS1 mice, spe-

Woolley et al., 1990), and the composition of the synaptic proteomecifically in the microglial population, but not in neurons (Figure 7f

varies between male and female mice (Distler et al., 2020). Many ofand Figure S1d). We conclude that the sex-­specific difference inthe sex differences in structural plasticity are driven by hormones,

FcγRIIb expression identified via mRNAseq is attributed primarily toand hence, their influence is more pronounced in females (Hyeran increase in AD pathology driven by activation of microglia.

et al., 2018). For example, oestrogen increases spine density in an

F I G U R E 7 APP/PS1 female mice have more Aβ plaques and microglia. (a) Hippocampal slices from APP/PS1 mice from both sexes were
processed and immunolabelled with antibodies against Iba-­1 (green), Aβ (red) and Hoechst nuclear dye (cyan; scale bar: 200 µm). The whitebox shows a magnified region of the CA1 cell body layer (right most panels; scale bar: 20 µm). For analysing immunolabelled structures, a
total of four non-­overlapping hippocampal coronal sections per animal were quantified (labelled as different shapes), three animals per groupwere imaged with a slide scanner and quantified. Graphs show Aβ plaque burden (b) and total microglia counts (d) normalized over area of
measurement, as well as cumulative frequency of plaques sizes measured across all slices (males, n = 6060; females, n = 7163); (c). The percent change in plaque burden and microglia between male and female mice for each animal (n = 3) was also quantified and graphed (e). (f)Hippocampal slices were immunolabelled with antibodies against Iba-­1 (green), NeuN (purple), FcγRIIb (red) and nuclear dye, Hoechst (cyan).
The Iba-­1-­positive FcγRIIb signals were quantified for each slice and plotted on the bar graph (scale bar: 200 µm). Statistical analyses of allgroup data were performed using unpaired Student's t test with Welch's correction (****p<0.0001, ***p<0.001 and **p<0.01)

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NAVAKKODE et al.

extracellular signal-­regulated protein kinase (ERK)-­dependent man-guidance but also regulates dendrite branching and spine matura-ner and regulates gene transcription and protein synthesis (Nilsson

tion (Morita et al., 2006). Mef2c is a well-­known activity-­dependent

et al., 2001). Ovariectomized female rodents also displayed a declinetranscription factor involved in learning and memory (Barbosa et al.,in spine density that is reversed by hormonal supplements (Gould

2008). In addition, three of the female-­specific DEGs that had re-et al., 1990; MacLusky et al., 2005). LTP in the Schaffer collateral

duced expression in APP/PS1 mice, including Syt17, are involved inpathway is also modulated by oestrogen and expression of endoge-

AMPAR trafficking during synaptic plasticity, suggesting this func-

nous estrogen receptor α (ERα) in females, but not in males. Infusion

tion may be disrupted. Intriguingly, expression of both Mef2c andof oestradiol facilitated LTP and synaptic signalling by ERα and ERβ in

Syt17 has been shown to be affected by androgens in neurodevel-

females, but not in males (Wang et al., 2018). Moreover, activation of

opment, suggesting that the effect of sex hormones on plasticity

kinases such as Src, ERK and TrkB is modulated by expression of ERα

gene expression may be a key factor predisposing females to AD

in females (Wang et al., 2018). Given the extensive influence of hor-

(Lombardo et al., 2020). Future studies targeting differentially ex-mones on neural plasticity, any hormone imbalance or disruption inpressed genes in female mouse models for AD that are independent

hormone-­responsive plasticity mechanisms in AD conditions couldof the immune system but associated with neuronal plasticity might

explain the faster decay of LTP and decline in associative memories

prove to be informative.

in AD females (Lu et al., 2019). While sex hormones decline during

The brain slice immunostaining validates our gene expression

normal ageing, in AD, there is evidence that brain levels of sex hor-data, which points to females showing advanced Aβ pathology ac-

mones (Rosario et al., 2004, 2011; Yue et al., 2005) along with sex

companied by a stronger inflammatory response. This accelerated

hormone receptor expression, function and subcellular distribution

progression has been reported in other mouse models and in AD pa-

are further compromised (Long et al., 2012; Lu et al., 2003). Whiletients (Wang et al., 2020; Yang et al., 2018). As previously discussed,

the relationship between hormones and its impact on AD remains

sex differences in synaptic plasticity may be responsible for synaptic

to be clarified, altered brain hormone levels and its decline in re-dysfunction in AD, but the accelerated pathology and the increased

sponsiveness in AD may provide clues in understanding sex-­specificactivation of microglia can also contribute towards impairments indifferences in impaired neural plasticity and memory loss.

neural plasticity. Microglia are essential for synaptic pruning, andIt should be noted that the oestrous cycle of female animals did

aberrant microglial-­mediated engulfment of synapses in response to

not impact our LTP recordings, as we did not find any significant

elevated Aβ plaque levels could conceivably impact synaptic trans-differences in potentiation and decay across the various oestrous

mission (Hong et al., 2016; Parihar & Brewer, 2010; Raghuraman

cycles (Figure S2). The involvement of the oestrous cycle towards

et al., 2019). Alternatively, elevated Aβ levels in the extracellularsynaptic plasticity and LTP remains unresolved with some studies

microenvironment can also directly impact synaptic efficacy. For ex-

reporting enhancement of CA1 LTP during specific pro-­oestrous

ample, Aβ plaques are known to disrupt the synthesis of plasticity-­

periods while others showing no significant differences in CA1 LTP

related proteins required for the maintenance of LTP (Sharma et al.,(Hong et al., 2016; Warren et al., 1995).

2017). Thus, a higher Aβ plaque burden might account for the lower

Our transcriptome profiles of the male and female APP/PS1

expression of plasticity proteins. Interestingly, FcγRIIb expression

hippocampus indicated an overall increase in gene expression as-

has been reported not only in microglia but also in circulating mac-sociated with the immune response. Many of the canonical markers

rophages and other myeloid populations, raising the possibility thatfor microglial activation and neuroinflammation were upregulated

the increased expression in female AD mice could also be attributedin both sexes, while female mice displayed a twofold increase into the infiltration of circulating macrophages (Quan et al., 2009).

differentially expressed genes compared with their male counter-

Earlier studies have shown that soluble Aβ42 oligomers signifi-

parts, suggesting a strong sex-­specific difference in response tocantly blocked the induction and maintenance of HFS-­LTP, but not

AD pathology. While slice recordings indicated that both males andTBS-­LTP, thus showing that sex difference depends on the stimula-females have impaired long-­term plasticity, it was surprising to seetion protocol (Smith et al., 2009). Sex difference in the amplitude ofthat only female mice showed a broad downregulation of genes as-

LTP also depends on hippocampal projections, as a robust sex dif-sociated with plasticity and memory. Collectively, these changes inference in the magnitude of LTP was observed in DG after perforantgene expression may account for the accelerated decay of late LTP

path stimulation (Maren, 1995). Similar findings were also observed

and STDP. It should be noted that while the expression of plasticity

from timing-­induced plasticity, as pSTDP was impaired in both malegene sets was overall reduced in APP/PS1 females, we detected rel-and female hippocampal CA1 regions with female APP/PS1 mice

atively few changes in individual genes. This could reflect a grad-showing a more prominent decay. Similarly, a study in human AD

ual dysregulation of diverse molecular processes associated withpatients failed to induce STDP in the cortico-­cortical connections

neuronal plasticity rather than single-­gene disruptions in APP/PS1(Di Lorenzo et al., 2018). Also consistent with our findings, Garadfemale mice. In addition, we also identified a set of female-­specific,

and colleagues reported that STDP in APP/PS1 mice was impaired

differentially expressed neuronal genes previously linked to AD

in an Aβ plaque distance-­dependent manner (Garad et al., 2021),

pathology including Sema3a and Mef2c (Abad et al., 2006; Liangsuggesting that a higher Aβ load in female APP/PS1 mice might be

et al., 2010). Both genes are known regulators of neuronal plasticity.

a reason for the faster decay we observed in slice physiology and inSema3a not only encodes a secreted protein that is critical for axon

the BT paradigm.

| 17 of 21

NAVAKKODE et al.

In agreement with our LTP and STDP findings, our behavioural

AC K N OW L E D G E M E N T S

experiments also revealed that APP/PS1 female mice are able to

The authors thank Prof. George J. Augustine, Lee Kong Chian Schoolacquire IA memory, while they show impairment in the formationof Medicine, Nanyang Technological University, Singapore, for his

of IA-­LTM results. Previous studies have already shown that APP/

insightful comments on this manuscript and Ms. Ang Ruixia Sheila

PS1 mice have long-­term spatial memory deficits (Krishna-­K et al.,for the help with text editing the manuscript. This research is sup-2020), but in order to uncover sex-­s pecific impairment in associa-

ported by Singapore Ministry of Education Academic Research Fundtive memories, we employed the BT paradigm, that is known toTier 3 (MOE2017-­T3-­1-­0 02; THC and SS) and by Nanyang Assistantcapture subtle behavioural differences that are normally masked

Professorship (NAP) from Nanyang Technological University,

in standard memory tasks (Gros & Wang, 2018). We proposed

Singapore. A part of this work was also supported by NUSMED-­FOS

earlier that the deficit in associative memory in APP/PS1 mice

Joint Research Programme (NUHSRO/2018/075/NUSMed-­FoS/01)

may occur due to altered expression of plasticity-­r elated proteinsto S.S.

(PRPs) or impaired setting of synaptic tags in neurodegenerative
neural networks. We found that plasticity-­r elated genes wereC O N FL I C T O F I N T E R E S T

downregulated in females compared with male APP/PS1 mice.

The authors declare that they have no conflict of interest.

Moreover, the “tag” setting processes seemed not to be impaired
because STM was intact in all groups. However, we cannot rule

AU T H O R C O N T R I B U T I O N S

out the possibility that STM can be sustained by other molecu-

SN, JRG, THC and SS conceived and coordinated the study. SN,

lar/cellular processes that do not include tag setting. In line with

JRG, THC and SS wrote the paper. SN, MVP and RPA performed

that, Gros and Wang reported that novelty did not facilitate long-­

and analysed the electrophysiology experiments. JRG and VB per-term memory persistence in middle-­a ged rats [i.e., behaviouralformed and analysed the transcriptome and immunohistochem-

tagging was impaired] and they concluded that this deficit wasistry experiments. SN performed and analysed the behaviouralmost likely due to impairments in tag setting rather than PRP syn-experiments. YSC performed dissections for immunohistochem-thesis in early ageing (Gros & Wang, 2018). In our studies, we

istry experiments.

observed that APP/PS1 mice were able to acquire memories, revealing the ability to set learning tag, while impairment of asso-

DATA AVA I L A B I L I T Y S TAT E M E N T

ciative memory suggests a reduction in the availability of PRPs.

All RNAseq datasets can be uploaded from NCBI GEO: GSE186710.

Nevertheless, APP/PS1 mice had significantly shorter latencies at
all key time points, indicating that tag setting process may be im-

ORCID

paired partially. Our results show enhanced microglial activation

Sheeja Navakkode

in AD mice, females in particular are strengthened by our earlier

Toh Hean Ch’ng

finding showing the specific role of microglia in tag setting andSreedharan Sajikumar

https://orcid.org/0000-0003-1754-711X
https://orcid.org/0000-0002-7170-1512
https://orcid.org/0000-0002-5761-8982

STC (Raghuraman et al., 2019). We also observe a faster decay in
LTP in APP/PS1 female mice, which suggests the possibility that

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How to cite this article: Navakkode, S., Gaunt, J. R., Pavon,
M. V., Bansal, V. A., Abraham, R. P., Chong, Y. S., Ch'ng, T. H.,& Sajikumar, S. (2021). Sex-­specific accelerated decay intime/activity-­dependent plasticity and associative memory inan animal model of Alzheimer's disease. Aging Cell, 20,
e13502. https://doi.org/10.1111/acel.13502